Because fluorescent materials (phosphors) obtained by dispersing rare-earth ions, transition metal ions, and the like in inorganic materials have better durability than organic dyes, these fluorescent materials have been conventionally used for lights, displays, and the like. However, because the brightness and color-rendering properties thereof are not always sufficient, there has been a demand for a fluorescent material with higher brightness. In recent years, semiconductor nanoparticles (particle size of several nanometers; without doping of rare-earth ions or transition metal ions; hereinafter also simply referred to as “nanoparticles” or “quantum dots”) are gaining attention as a high-performance fluorescent material that embodies the above demand because of the following reasons: semiconductor nanoparticles are excellent in color-rendering properties because these particles emit bright fluorescence of various wavelengths according to the particle size even when irradiated with ultraviolet light of the same wavelength, and the brightness of these nanoparticles can be increased because their emission decay time is short. If semiconductor nanoparticles are carefully prepared, the brightness becomes high to the degree that the emission of each particle can be separately detected and spectroscopically analyzed. Consequently, in addition to the use for displays and light, there is the beginning of a great development in the field of application where semiconductor nanoparticles are conjugated to biomolecules and used as fluorescent probes for the elucidation of the mechanism of the life, the diagnosis of diseases, and the like.
Primary examples of semiconductors that serve as the above-described fluorescent materials include II-VI semiconductors (cadmium sulfide (CdS), zinc selenide (ZnSe), cadmium selenide (CdSe), zinc telluride (ZnTe), cadmium telluride (CdTe), mixed crystals thereof, etc.) and III-V semiconductors (indium phosphide (InP), etc.). These are direct transition semiconductors, and their emission lifetime is about 10 nanoseconds, which is about five orders of magnitude smaller than conventional forbidden transition fluorescent materials that use a rare-earth ions or transition metal ions. Consequently, fluorescence with much higher brightness can be achieved.
There are two synthesis methods for semiconductor nanoparticles that emit such high-intensity light (fluorescence): one method for synthesizing in an aqueous solution (hydrophilic nanoparticles are synthesized), and another method for synthesizing in an organic solution (non-polar solvent) in which water is removed at a high level (hydrophobic nanoparticles are synthesized). Because nanoparticles have a large specific surface area, these nanoparticles are gradually agglomerated in the solution in order to reduce the surface energy, and the PL efficiency is thus decreased. Therefore, there was a problem with the nanoparticles synthesized by both methods in that it was difficult to put them to practical use when these nanoparticles were in the form of a solution. In order to solve this problem, semiconductor nanoparticles must be incorporated in a transparent matrix in such a manner that the nanoparticles are dispersed and fixed therein so as to obtain a solid material that maintains the initial properties for a long period of time under various environments. As a solid matrix therefor, there are two materials: glass, and organic polymer materials. Between these, glass, particularly silica glass, has a higher transparency and a higher tolerance to ultraviolet irradiation than organic polymers. Additionally, moisture and oxygen cannot easily permeate through silica glass when silica glass is formed in a network structure, making it possible to prevent degradation of dispersed nanoparticles for a long period of time. A sol-gel method is favorable for the preparation of such glass because in the sol-gel method, vitrification progresses under mild conditions at or close to normal temperature and pressure; thus, if a preparation method is improved, semiconductor nanoparticles can be dispersed and fixed in a transparent glass while maintaining high PL efficiency that was achieved immediately after synthesis by the solution method. Because the sol-gel method uses water, it is preferable to use hydrophilic nanoparticles from the viewpoint of preventing agglomeration and quenching.
The term “silica glass” is explained here. Although glass prepared by the sol-gel method contains an organic materials and water, such a product is called glass, silica, silica glass, silica-based glass, amorphous silica, SiO2, etc., in related scientific societies. This is because other metal ions that modify the network structure formed by silicon are not contained in the prepared solid matrix. Therefore, a matrix containing silicon prepared by the sol-gel method is also called glass, silica, or silica glass in the present description.
The present inventors developed a bulk-like glass (Patent Literature 1), glass fine particles (Patent Literature 2, 3, and 4), and a glass thin film (Patent Literature 5) as the fluorescent glass described above. Of these, glass fine particles (particle size of 10 nm to 2 μm; when the particle is not a complete sphere, for example, a rugby ball shape (spheroid elongated in the direction of the symmetrical axis), pancake shape (flattened spheroid), and the like, the average length of three principal axes of inertia is defined as the particle size in the present description; when the particle is a complete sphere, the diameter is the particle size) can be used as powdered fluorescent material for light-emitting devices such as displays, lights, and the like. In addition, such glass fine particles have an important application as fluorescent probes by being bound to biomolecules. The description is given below, by limiting to the fluorescent silica glass fine particles.
In Patent Literature 2, 3, and 4 of the present inventors, the sol-gel method in which alkoxide is hydrolyzed and dehydration-condensed is used. In particular, a reverse micelle method (a method in which the sol-gel method is performed in minute water droplets dispersed in the oil phase, wherein water-dispersible nanoparticles are dispersed in the water droplets in advance) or Stöber method (a method in which hydrolyzed alkoxide is deposited on the nanoparticle surface) was used to develop a technology to disperse multiple semiconductor nanoparticles in silica glass fine particles with high PL efficiency (25% or higher).
However, when the prepared silica glass fine particles are applied as fluorescent probes in the field of biotechnology, conditions for the evaluation of fluorescence properties are usually significantly different from when the particles are prepared as usual phosphor such as for light-emitting materials.
Because silica glass fine particles have less scattering when the particle size is about 100 nm or less, the particles are introduced into a quartz cell having a light path of 1 cm while being dispersed in a solution, and the quartz cell is measured using an absorption spectrophotometer and a fluorescence spectrophotometer for general purposes. Thereby, the absorbance and the fluorescence intensity of each wavelength are determined. An integrating sphere is used when the influence of scattering is a concern; however, in this case, there is an increase in errors in both absorbance and fluorescence intensity, compared to the case where there is no scattering. Also in this case, general-purpose measurement devices have recently been commercially available (for example, C9920-02 by Hamamatsu Photonics K.K.).
The concentration of semiconductor nanoparticles just after synthesis is usually about 1 to about 10 μM (μmol/L; this indicates the number of semiconductor nanoparticles, rather than the number of atoms constituting the nanoparticles). These particles are stored as-is in a cool, dark space. When measuring the PL efficiency, the concentration is diluted to about 200 to about 300 nM because the above concentration is too high. Consequently, the signal level that can be most easily measured using a general-purpose absorption spectrophotometer or fluorescence spectrophotometer is obtained. Pure water is often used as the solvent. On the other hand, when semiconductor nanoparticles are applied as fluorescent probes, the fluorescence is often separately detected from one or several nanoparticles; and in that case, the nanoparticle concentration is about 10 nM at most, and the nanoparticles are dispersed in a highly concentrated salt solution such as saline. Further, the irradiated light intensity is also usually 10 W/cm2 or greater, which is more intense compared to irradiated light intensity when measuring using a spectrometer by a different order of magnitude. In this way, it became clear that, in terms of material synthesis as described above, when the concentration of dispersed nanoparticles is extremely low and a large amount of salts are contained in the solution, there is a case where nanoparticles are degraded even if glass is used for coating of the nanoparticles. In order to prevent such degradation, developing a glass network structure is one effective means. Further, it is more preferable, from the viewpoint of increasing the brightness, to incorporate multiple nanoparticles in glass to form one glass fluorescent fine particle than to coat glass with one nanoparticle.
As described later, the present inventors found that, among the nanoparticles described in Patent Literature 2 to 4 above, it is effective to use nanoparticles containing Cd and Se, for example, CdSe nanoparticles, in order to prevent degradation. However, CdSe nanoparticles having high PL efficiency are prepared by an organic solution method in which water is removed at a high level, and the CdSe nanoparticles are quenched when they are dispersed as-is in an aqueous solution. Therefore, it is desirable to disperse CdSe nanoparticles in glass fine particles while maintaining the PL efficiency thereof. In particular, it is desirable to disperse numerous nanoparticles in order to increase the brightness as much as possible.
Further, in the search of a possible application in the field of biotechnology, it became clear that fluorescent fine particles having a particle size of 100 nm or less are effective. Cells are typically 10 to 30 μm in size, and when the particle size exceeds 100 nm, the possibility of the particles being internalized by cells through phagocytosis decreases. Further, when the cell interior is stained in various colors, the particle size of over 100 nm and close to 200 nm is not suitable for clear staining, because the shape thereof can be seen under the optical microscope when the particle size is in that range. On the other hand, the particle size of semiconductor nanoparticles is a few to several nanometers, and fine particles in which 10 or more nanoparticles are dispersed are necessary in order to increase the brightness by a different order of magnitude. In order to do so, the particle size must be about 20 nm or more.
Next, the present inventors examined particles known as silica glass fine particles in which nanoparticles containing Cd and Se are dispersed, and the emission properties thereof.
Bawendi et al. reported fluorescent silica glass fine particles in which CdSe/ZnS nanoparticles are dispersed and fixed in glass by the sol-gel method, and a method for preparing the particles (Non-Patent Literature 1). This preparation method is a method in which the surface of nanoparticles that have been synthesized in an organic solvent in advance is coated with alkoxide having an amino group (3-aminopropyltrimethoxysilane) and alcohol having an amino group (5-amino-1-pentanol), and the resulting product is bound as a layer having a thickness of about 50 nm to the surface of separately prepared silica glass fine particles having a diameter of about a few hundred nanometers. This method provides fluorescent glass fine particles having a structure in which the surface of silica glass fine particles not containing nanoparticles is coated with a sol-gel glass layer containing nanoparticles. However, because the nanoparticles are present only around and on the surface of the glass fine particles, and no nanoparticles are contained in the core of the glass fine particles, it was not possible to increase the concentration of dispersed nanoparticles in the glass fine particles. Additionally, the PL efficiency was about 13%.
As another preparation method, a method in which alkoxide having a thiol group or the like is formed on the surface of CdSe/ZnS nanoparticles, and silica glass fine particles containing one nanoparticle per one silica glass fine particle are prepared (Non-Patent Literature 2) has been reported. The PL efficiency in this case is reported to be 5 to 18%. There is a report on a silica glass fine particle prepared by a similar method, wherein the particle has a particle size of 30 nm to 1 nm and contains one CdSe/ZnS nanoparticle; however, this report is silent about the PL efficiency (Non-Patent Literature 3).
Meijerink et al. introduced CdSe/CdS/Cd0.5Zn0.5S/ZnS (CdSe as the core is sequentially coated with CdS, Cd0.5Zn0.5S, and ZnS) nanoparticles into silica glass fine particles by a reverse micelle method in order to introduce one nanoparticle into one silica glass fine particle. However, based on the examination of the mechanism, it was found that because hydrolyzed alkoxide has a high affinity for nanoparticles, the ligands arranged on the nanoparticle surface at the time of preparation are replaced by the hydrolyzed alkoxide, thus quenching the emission. Accordingly, in regard to the silica glass fine particle containing only one nanoparticle, the PL efficiency of the nanoparticle was rapidly decreased immediately after preparation, and was further gradually decreased. One week after preparation, the PL efficiency was about 2% of what it was before being introduced into the silica glass (a drop from the initial value of 60% to 1.2% in the absolute value). In order to suppress such quenching effect of silica glass, a nanoparticle with a specially made thick shell was used. As a result, the PL efficiency was increased to a maximum of 35% (Non-Patent Literature 4). However, such a nanoparticle with a specially made thick shell has a large particle size, and is not suitable for application in the field of biotechnology; additionally, it is difficult to prepare such nanoparticles.
There is known research in which a water-dispersible CdSe nanoparticle (citric acid coating) was prepared, and several of these nanoparticles were introduced into silica glass fine particles. However, the PL efficiency of water-dispersible CdSe nanoparticles is 0.1 to 0.15%, which is extremely low (Non-Patent Literature 5). The PL efficiency of the nanoparticle when it is introduced into a silica matrix is nowhere described; however, the PL efficiency is usually further decreased in that case. Therefore, such a product cannot be called a “fluorescent material (phosphor).” Further, in the case of relatively recent literature (Non-Patent Literature 6) in which a water-dispersible CdSe nanoparticle was similarly introduced into a silica particle by a reverse micelle method, the PL efficiency was 1.48% at most, and this nanoparticle cannot be called a “fluorescent material (phosphor).” As illustrated in Patent Literature 6, as a rough standard, the PL efficiency should be 20% or higher for a nanoparticle to be called a fluorescent material (phosphor).
As described above, a method for preparing fluorescent silica fine particles having a particle size of 20 to 100 nm, in which 10 or more nanoparticles containing Cd and Se are dispersed, has not been developed.
Meanwhile, recently, there is a report on a method for preparing an assembly of multiple nanoparticles using a linear polymer (Non-Patent Literature 7). Polymer particles prepared by this method are reported to have an average particle size of 112 nm, as measured by dynamic light scattering. At present, it is difficult to prepare nanoparticles having a particle size of 100 nm or less. It is possible to glass-coat the surface of the assembly; however, this further increases the particle size. Further, a glass material not containing a polymer generally has better durability, and produces a smaller amount of dissolved Cd released from particles. Therefore, there is a demand to prepare a glass material in which numerous semiconductor nanoparticles are dispersed, without using a polymer.